Replacing a piece of loop-structure in the substrate-binding groove of Aspergillus usamii beta-mannanase, AuMan5A, to improve its enzymatic properties by rational design

摘要

To perfect the enzymatic properties of AuMan5A, a mesophilic glycoside hydrolase (GH) family 5 beta-mannanase from Aspergillus usamii, its loop-structure substitution was carried out by rational design and followed by megaprimer PCR. Based on the structural analysis and enzymatic property comparison of various beta-mannanases, a piece of loop-structure with seven amino acids between two beta-strands (beta D and beta E) in the substrate-binding groove, named "Loop DE," was speculated to be correlative to the thermostability and catalytic efficiency of GH family 5 beta-mannanases. Therefore, three AuMan5A's mutants, AuMan5A-Af, AuMan5A-An, and AuMan5A-Th, were designed by substituting a Loop DE sequence ((316)KSPDGGN(322)) of AuMan5A with the corresponding sequences of other three family 5 beta-mannanases, respectively. Then, the mutant-encoding genes, Auman5A-Af, Auman5A-An, and Auman5A-Th, were constructed as designed theoretically and then expressed in Pichia pastoris GS115. The expressed recombinant AuMan5A-Af (re-AuMan5A-Af) displayed the temperature optimum (T (opt)) of 75 A degrees C, T (m) value of 76.6 A degrees C and half-life (t (1/2)) of 480 min at 70 A degrees C, which were 10 and 12.1 A degrees C higher and 48-fold longer than those of re-AuMan5A, respectively. Its catalytic efficiency (k (cat)/K (m)) was 12.7-fold that of re-AuMan5A. What is more, the site-directed mutagenesis of D320G in AuMan5A-Af was performed. The T (opt) and t (1/2) of expressed re-AuMan5A-Af(D320G) decreased to 70 A degrees C and 40 min, respectively, while its k (cat)/K (m) was only 35 % of that of re-AuMan5A-Af. These results demonstrated that the mutation of G320 (in AuMan5A) into D320 (in AuMan5A-Af) through Loop DE substitution was mainly responsible for the thermostability and catalytic efficiency improvement of AuMan5A-Af.